The Ability of Saprotrophic Bacteria Isolated from Natural Habitats to Lyse Yeasts

More than 600 bacterial strains isolated from different horizons of steppe biogeocenoses and zoogenous loci (diplopod intestines and feces) were tested for the ability to lyse yeast cell walls. About half of the strains that were isolated from biotopes with active degradation of plant debris (steppe litters and diplopod intestines and feces) were found to possess yeast-lytic activity. Most of the yeast-lytic strains belonged to the genera Streptomyces, Promicromonospora, Oerskovia, and Agromyces. The yeast-lytic activity of actinobacteria from the genera Agromyces, Mycobacterium, and Micrococcus has not previously been reported.     

Heat Resistance and α-Toxigenicity of Clostridium perfringens Strains in Normal Intestines of Japanese

Heat-resistant strains of Clostridium perfringens were isolated from normal feces samples of Japanese people more frequently than from European people. However, the predominant number of the C. perfringens cells in the human intestines were sensitive to the heat at 100 C for 10 min. This proved to be true of the feces samples which were demonstrated to carry the organism resistant to 100 C for 60 min. Further studies indicated that the more the number of the organism in the intestines, the higher was the recovery ratio of the heat-resistant strains. Concomitant estimation for α-toxigenicity of those strains isolated from unheated feces samples revealed that the α-toxigenicity ranged mainly between 0.05 and 0.9 α-antitoxin equivalents.     

6-Aminohexanoate oligomer hydrolases from the alkalophilic bacteria Agromyces sp. strain KY5R and Kocuria sp. strain KY2

Alkalophilic, nylon oligomer-degrading strains, Agromyces sp. and Kocuria sp., were isolated from the wastewater of a nylon-6 factory and from activated sludge from a sewage disposal plant. The 6-aminohexanoate oligomer hydrolases (NylC) from the alkalophilic strains had 95.8 to 98.6% similarity to the enzyme in neutrophilic Arthrobacter sp. but had superior thermostability, activity under alkaline conditions, and affinity for nylon-related substrates, which would be advantageous for biotechnological applications.     

Noninvasive Analysis of Microbiome Dynamics in the Fruit Fly Drosophila melanogaster

The diversity and structure of the intestinal microbial community has a strong influence on life history. To understand how hosts and microbes interact, model organisms with comparatively simple microbial communities, such as the fruit fly (Drosophila melanogaster), offer key advantages. However, studies of the Drosophila microbiome are limited to a single point in time, because flies are typically sacrificed for DNA extraction. In order to test whether noninvasive approaches, such as sampling of fly feces, could be a means to assess fly-associated communities over time on the same cohort of flies, we compared the microbial communities of fly feces, dissected fly intestines, and whole flies across three different Drosophila strains. Bacterial species identified in either whole flies or isolated intestines were reproducibly found in feces samples. Although the bacterial communities of feces and intestinal samples were not identical, they shared similarities and obviously the same origin. In contrast to material from whole flies and intestines, feces samples were not compromised by Wolbachia spp. infections, which are widespread in laboratory and wild strains. In a proof-of-principle experiment, we showed that simple nutritional interventions, such as a high-fat diet or short-term starvation, had drastic and long-lasting effects on the micobiome. Thus, the analysis of feces can supplement the toolbox for microbiome studies in Drosophila, unleashing the full potential of such studies in time course experiments where multiple samples from single populations are obtained during aging, development, or experimental manipulations.     

胆盐水解酶产生菌的筛选及其益生潜力研究

目的从健康仔猪肠道及粪便中筛选具有胆盐水解酶活性的优良益生乳杆菌菌株,并对筛选菌株的体外、体内益生潜力进行初步探讨。方法用MRS培养基分别从仔猪新鲜粪便和小肠黏膜分离培养乳酸菌,用筛选鉴别培养基筛选阳性菌株,研究菌株的抗逆能力、黏附特性以及体内益生活性。结果从粪便和小肠黏膜共得到乳酸菌388株和97株,其中胆盐水解酶阳性菌株分别为291株(75%)和86株(89%)。选择5株胆盐水解酶活性最强、能水解游离胆酸的菌株,研究菌株的抗逆能力、黏附特性以及体内益生活性,并对最终选育得到的菌株进行生理生化及16S rRNA分子鉴定,发现菌株罗伊乳杆菌G22-2能耐受pH 3.0的酸度、1.0%的胆盐浓度,在小肠上皮细胞上的黏附效率超过15个以上;通过大鼠体内实验,筛选出的菌株G22-2对大鼠无毒无害,并能显著改善血脂水平。结论罗伊乳杆菌G22-2符合益生菌的要求,可以作为产胆盐水解酶的益生乳杆菌优良的候选菌株。     

Ruminant feces harbor diverse uncultured symbiotic actinobacteria

To isolate actinobacteria from ruminant feces and elucidate their correlations with ruminants, the actinobacterial community in sheep (Ovis aries) and cattle (Bos taurus) feces was determined by cultivation and clone library methods. Most of actinobacteria isolated belonged to Streptomyces, Amycolatopsis, Micromonospora, and Cellulosimicrobium genera. The strains showed above 99 % similarity with the type strains, respectively. All the strains isolated could grow on media containing pectin, cellulose, or xylan as the sole carbon sources. However, most antibacterial and antifungal activities were found in Streptomyces species. Clone library analysis revealed that the genera Mycobacterium, Aeromicrobium, Rhodococcus, Cellulomonas were present in cattle and sheep feces. In contrast, the 16S rRNA genes showed less than 98 % similarity with the type strains. The analysis of actinobacterial community in ruminant feces by clone library and cultivation yielded a total of 10 actinobacterial genera and three uncultured actinobacterial taxa. The ruminant feces harbored diverse actinobacterial community. Ruminants may represent an underexplored reservoir of novel actinomycetes of potential interest for probiotics and drug discovery.     

产淀粉酶益生乳杆菌的筛选及鉴定

目的以健康仔猪肠道及粪便样品为基础,从中筛选产淀粉酶的乳杆菌菌株,并评价其作为益生菌候选菌株潜力。方法用MRS培养基分别从仔猪新鲜粪便和小肠黏膜上分离到乳酸菌,采用改良的产淀粉酶选择性培养基初筛得到能降解淀粉活性的菌株,并研究菌株的淀粉水解活性、抗逆能力、粘附特性及对抗生素的敏感性。结果从备选的485株乳酸菌中筛选得到具有初步淀粉酶活性的菌株25株（占总筛选数量的5.2%）,复筛选育得到具有较强淀粉酶活性的乳杆菌3株。进一步研究了这3株乳杆菌的抗逆能力、粘附特性以及对抗生素的耐药性,并对最终选育得到的菌株进行生理生化及16S rRNA分子鉴定。经选育鉴定的罗伊乳杆菌G8-5淀粉降解能力最强,并能耐受pH 3.0的酸度、1.0%的胆盐浓度,在小肠上皮细胞上的粘附效率超过15个以上,并对常用的抗生素具有较高的敏感率。结论罗伊乳杆菌G8-5符合安全益生菌的要求,可以作为产淀粉酶的益生乳杆菌优良的候选菌株。     

几株细菌的重金属抗性水平和吸附量

从堆积时间为80～100a的铅锌矿渣中分离了6株细菌,通过测定部分16S rRNA基因序列确定了它们的系统发育地位。结果表明有3株细菌属于节杆菌属(Arthrobacter),同A．nicotinovorans和A．histidinolovorans两个种关系密切。另外3株属于壤霉菌属(Agromyces),同Ag．mediolanus具有较近的亲缘关系。总体来看,这些菌株都对检测的5种重金属有高的最低抑菌浓度(minimal inhibitory concentration,MIC)。节杆菌对Zn、Co的耐受明显高于壤霉菌。此外,这些重金属高抗性菌株也对重金属有较强的吸附能力。在环境中有单一重金属离子的情况下,冻干的节杆菌对Ph的吸附率平均达到了约400mg／g干菌体,对Cd和zn的吸附也分别达到了近177和80mg/g干菌体,具有进一步开发为重金属吸附剂的潜力。     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Extracellular yeast-lytic enzyme of the bacterium <Emphasis Type="Italic">Lysobacter</Emphasis> sp. XL 1

An enzyme exhibiting yeast-lytic activity has been isolated from the culture liquid of the bacterium Lysobacter sp. XL 1. The optimal conditions for the hydrolysis of Saccharomyces cerevisiae cells by the enzyme have been established: 0.15 M sodium acetate buffer, pH 6.0, 50 degrees C. The yeast-lytic activity of the enzyme is inhibited by EDTA, p-chloromercuribenzoate, and phenylmethylsulfonyl fluoride. According to the data of SDS-PAGE, the molecular weight of the protein is 36 kD. The enzyme hydrolyzes casein, hemoglobin, and synthetic peptide Abz-Ala-Ala-Phe-pNA, i.e. it exhibits proteolytic activity. The properties of the enzyme and its molecular weight correspond to those of a previously isolated extracellular metalloproteinase. The N-terminal amino acid sequence of the protein exhibits 67% homology with the N-terminal sequence of achromolysine of Achromobacter lyticus (EC 3.4.24.-).     

Geomicrobiological Study of the Grotta dei Cervi,Porto Badisco,Italy

Speleothems (active stalactites, wall concretions), rock walls, ceiling, and soils from the galleries of Grotta dei Cervi, Porto Badisco, Italy, were sampled to investigate the culturable heterotrophic microbial communities present in this cave. Sampling was carried out in a transect of about 230 m from the entrance to the central gallery where numerous Gram-positive bacteria were isolated from all studied sites. Members of the genera Agromyces, Arthrobacter, Rhodococcus , and particularly Streptomyces , of the order Actinomycetales, are spread throughout the whole cave. The ability of actinomycetes, and particularly of Nocardiopsis , to colonize salt-stressed environments is favored by the presence of ectoine, a compatible solute for osmotic adaptation. Selected actinomycete isolates were tested for the formation of crystals. Strains from all tested genera, except isolates of Gordonia and Nocardia , produced vaterite and/or calcite. Production of Mgcalcite was restricted to strains of Brachybacterium, Rhodococcus , and Streptomyces , whereas struvite was only precipitated by an unidentified isolate. These findings indicate that actinomycetes may play a role in the formation of mineral deposits in caves.     

Formation of Cyclohexylamine and Cyclohexanone from Cyclamate by Microorganisms Isolated from the Feces of Guinea Pig

The assimilation of sodium cyclamate (CHS-Na) by microorganisms was studied. Fifteen strains of cyclamate-assimilating bacteria were isolated from the feces of guinea pig excreting cyclohexylamine (CHA) in urine. The majority of the strain isolated seems to belong to the genera Pseudomonas and Corynebacterium. All strains were able to assimilate CHS-Na as a sole source of carbon and nitrogen, and accumulated clearly CHA in the culture medium. It was confirmed with the cell-free extract that the strains possessed the enzyme system which formed cyclohexanone (CHnone) from CHS-Na via CHA. It seems that the desulfation of CHS-Na to CHA is catalyzed by hydrolase, and that the deamination of CHA to CHnone is catalyzed by amine oxidase depending on oxygen.     

Aggregation of lactobacilli and bifidobacteria with<Emphasis Type="Italic">Escherichia coli</Emphasis> O157

A total of 5 Bifidobacterium spp. isolated from pig and children' feces and 6 Lactobacillus spp. from chicken feces were examined for expression of aggregation, cell surface hydrophobicity (CSH) and adherence to intestinal mucin. Co-aggregation activity was seen in 3 strains of auto-aggregative bifidobacteria and 4 auto-aggregative strains of Lactobacillus spp. with 2 enterohemorrhagic E. coli (O157). CSH correlated with Lactobacillus auto-aggregating activity and adherence to mucin but the correlation between Bifidobacterium adherence to mucin and CSH was not confirmed.     

beta-Glucuronidase activities of intestinal bacteria determined both in vitro and in vivo in gnotobiotic rats.

The beta-glucuronidase activities of bacterial strains isolated from the rat intestinal tract were studied both in vitro in culture media and in vivo in the intestinal contents of gnotobiotic rats. Only 50 of 407 strains tested were found to be positive in vitro. They belonged to the three genera Clostridium, Peptostreptococcus, and Staphylococcus. The in vitro-negative strains were also negative in vivo. The beta-glucuronidase activities of the beta-glucuronidase activities of the positive strains were generally greater in vivo than in vitro. The highest in vivo activities were found in the intact bacterial cells and in the soluble fractions prepared from disrupted pellets. There was a discrepancy between the activities obtained from both conventional and gnotobiotic rats harboring selected positive strains, suggesting that the main beta-glucuronidase-positive strains have not yet been isolated from the intestines of conventional rats.     

beta-Glucuronidase activities of intestinal bacteria determined both in vitro and in vivo in gnotobiotic rats

The beta-glucuronidase activities of bacterial strains isolated from the rat intestinal tract were studied both in vitro in culture media and in vivo in the intestinal contents of gnotobiotic rats. Only 50 of 407 strains tested were found to be positive in vitro. They belonged to the three genera Clostridium, Peptostreptococcus, and Staphylococcus. The in vitro-negative strains were also negative in vivo. The beta-glucuronidase activities of the beta-glucuronidase activities of the positive strains were generally greater in vivo than in vitro. The highest in vivo activities were found in the intact bacterial cells and in the soluble fractions prepared from disrupted pellets. There was a discrepancy between the activities obtained from both conventional and gnotobiotic rats harboring selected positive strains, suggesting that the main beta-glucuronidase-positive strains have not yet been isolated from the intestines of conventional rats.     

Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.     

Microbiology of the stalactites from Grotta dei Cervi, Porto Badisco, Italy.

The active stalactites from Grotta dei Cervi, Porto Badisco, southeastern Italy, were sampled to investigate the microbial communities present in these speleothems. Sampling was carried out in a transect about 150 m long in the central gallery, where numerous Gram-positive bacteria were isolated. Actinomycetes of the genus Streptomyces were the most abundant, followed by members of the genus Bacillus. Further isolates were assigned to the genera Amycolatopsis, Arthrobacter; Agromyces. Micrococcus, Nocardiopsis and Rhodococcus of the order Actinomycetales. The ability of actinomycetes to colonize subterranean environments is discussed.     

Agromyces indicus sp. nov., isolated from mangroves sediment in Chorao Island, Goa, India

A Gram-positive, non-motile, rod-shaped bacterium strain NIO-1018(T) isolated from a mangrove sediment sample of the Chorao Island, Goa, India, was subjected to a detailed polyphasic taxonomic study. The strain designated as NIO-1018(T) matched with most of the phenotypic and chemotaxonomic properties of the genus Agromyces and represents a novel species. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NIO-1018(T) fell within the cluster comprising species of the genus Agromyces, clustering with Agromyces soli (98.1 %), Agromyces flavus (97.9 %), Agromyces aurantiacus (97.7 %) and Agromyces ulmi (97.3 %). The predominant menaquinone was MK-12, and the major cellular fatty acids were anteiso-C(15:0), iso-C(16:0), anteiso-C(17:0) and iso-C(15:0). The polar lipids consisted of diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G+C content of strain NIO-1018(T) was 71.8 mol%. The combination of phylogenetic analysis, DNA-DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain NIO-1018(T) represents a novel species of the genus Agromyces, for which the name Agromyces indicus sp. nov. is proposed. The type strain is NIO-1018 (=JCM 17573(T) = CCTCC AB2011122(T)).     

三江源地区不同退化程度草地的土壤放线菌区系

用不同分离方法,对三江源地区不同退化程度草地土壤放线菌的数量和多样性进行了比较.从5份土样中共分离放线茵178株,根据表型特征和16S rRNA基因序列分析,分别归入7个已知属:小单孢菌属(Micromonospora)、原小单孢菌属(Promicromonospora)、诺卡氏菌属(Nocardia)、假诺卡氏菌属(Pseudonocardia)、游动放线菌属(Actinoplanes)、韩国生工茵属(Kribbella)和链霉菌属(Streptomyces).其中链霉菌属分离菌株可归入7个表型类群.发现轻度退化高寒草原的土壤放线菌数量,丰度和多样性高于重度退化高寒草原;针茅高寒草原的土壤放线菌数量和多样性高于蒿草高寒草甸,而其中链霉菌的种类低于后者.表明高寒草地的退化程度与其中土壤放线茵的数量和多样性呈负相关.     

Isoprenoid pigments in representatives of the family Microbacteriaceae

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C40-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and beta-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.